Isolation of nucleic acids

2.2.1.1 Isolation of genomic DNA for genotyping of mice

To determine the genotype of mouse litters, a tail biopsy was taken at the age of three weeks. To isolate genomic DNA from mouse tail, it was proceded according to Wieczerzak (2012) with a modified centrifugation step. Therefore, 150 µl of direct lysis buffer (Peqlab) and 5 µl Proteinase K (10 µg/ml) were added and incubated overnight at 55 °C under shaking. Inactivation of proteinase K took place at 85 °C for 50 minutes and the samples were centrifuged at 13,000 rpm for 1 minute. Probes were kept at 8 °C until proceeding with the genotyping PCR using Immolase^TM DNA polymerase (compare 2.2.7).

2.2.1.2 Isolation of plasmid DNA from bacteria

The plasmid preparation is a procedure to isolate and purify plasmid DNA from bacteria. Three basic steps can be mentioned:

 preparation of a bacterial culture

 lyse the bacteria to extract plasmid DNA

 purification of plasmid DNA a) Mini-preparation

To extract and purify moderate yields of plasmid DNA from bacteria, the QIAprep^® Miniprep from Qiagen was used following the company’s instructions with some modifications regarding the amount of the buffers and incubation times. The principle of this procedure is based on a modified alkaline lysis method of Birnboim and Doly (1979). Starting material is a 5 ml overnight culture of E. coli in LB (Lysogeny Broth) medium with 5 µl of an appropriate antibiotic (50 mg/ml). First, the bacterial cells were pelleted by centrifugation at 4,000 rpm for 10 minutes at 8 °C. The supernatant was discarded and the pellet resuspended in 200 µl Buffer P1 (Resuspension Buffer) with RNase A (Qiagen protocol: 250 µl). Next, 200 µl Buffer P2 (Lysis Buffer) was added and mixed thoroughly by inverting the tube 4-6 times (Qiagen protocol: 250 µl). After incubation for 5 minutes at room temperature, 200 µl Buffer N3 (Neutralisation Buffer) was added (Qiagen protocol: 350 µl). The

44

solution was immediately mixed by inverting the tubes 4-6 times. Samples were incubated for 5 minutes on ice then centrifuged at 13,000 rpm for 20 minutes at 4 °C (Qiagen protocol: no incubation before the 10 minutes centrifugation step at room temperature). In this protocol no column is utilised; instead, the plasmid DNA is precipitated with isopropanol. The supernatant was carefully transferred to a new tube without taking debris from the pellet. To precipitate the DNA, 420 µl isopropanol was added, mixed and samples incubated for 15 minutes at room temperature before centrifuging at 13,000 rpm for 30 minutes at 4 °C. The supernatant was discarded and the DNA pellet washed with 500 µl 70 % ethanol.

After a final centrifugation step at 13,000 rpm for 5 minutes at 4 °C, the supernatant was discarded and the pellet air-dried. Depending on its size the DNA pellet was dissolved in 30-50 µl TE (Tris-EDTA) Buffer or ddH₂O. The DNA concentration was measured with the photometer (Eppendorf) (compare 2.2.2) and stored at -20 °C.

b) Midi-preparation

This method is used to isolate and purify plasmid DNA from bacteria. Here, the PureLink^® HiPure Plasmid Filter Purification Kit (for midi and maxi preparation of plasmid DNA) from Invitrogen was used which employs a patented anion-exchange resin that ensures high yields of highly pure plasmid DNA. The midiprep was performed according to the manufacturer’s instructions with slightly modifications.

First, the cell lysate was prepared. Departing from the manufacturer’s advice 30 ml instead of 15-25 ml of an overnight LB culture per sample was used for high copy number plasmids containing 30 µl of an appropriate antibiotic (50 mg/ml). The cells were harvested by centrifuging at 4,000 x g for 10 minutes at room temperature.

Next, 4 ml Resuspension Buffer (R3) with RNase A was added to the cell pellet which was vortexed until cells were homogenously resuspended (Invitrogen protocol: 10 ml R3 buffer). After that 4 ml Lysis Buffer (L7) was added and tubes were inverted until the lysate composite was homogenously mixed (Invitrogen protocol: 10 ml L7 buffer). Next, the lysate was incubated at room temperature for exactly 5 minutes. Then, 4 ml Precipitation Buffer (N3) was added and tubes were homogeneously mixed by inverting (Invitrogen protocol: 10 ml N3 buffer). While probes were centrifuging at 12,000 x g for 10 minutes at room temperature (not listed in the manufacturer’s protocol), the columns were equilibrated by applying 10 ml Equilibration Buffer (EQ1) directly to the filtration cartridge of the column

45

(Invitrogen protocol: 15 ml EQ1 buffer). After the solution drained by gravity flow, the precipitated lysate was transferred onto the column through a filter to prevent loading any protein remains. The column was washed twice with 10 ml Washing Buffer (W8). The buffer flew through the column by gravity flow until the flow stopped. The flow through was discarded and the DNA was eluted. Therefore, a sterile tube was placed under the column and 5 ml Elution Buffer (E4) was applied to the column. The solution was drained by gravity flow. Next, the DNA was precipitated by adding 3.5 ml isopropanol to the elution, then mixed well and incubated for 2 minutes at room temperature. A centrifugation step was performed at

> 12,000 x g for 30 minutes at 4 °C. The supernatant was removed and the pellet washed with 3 ml 70 % ethanol. After centrifuging at > 12,000 x g for 5 minutes at 4 °C, the supernatant was removed and the pellet was air-dried for about 10 minutes.

Depending on the pellet size the DNA was resuspended in 100-200 µl TE Buffer or ddH₂O. The concentration of the precipitated DNA was measured with a spectral photometer (Eppendorf) (compare 2.2.2) and stored at -20 °C.

2.2.1.3 RNA isolation from mouse embryos

For the RNA isolation E9.5 old Whirligig mouse embryos (wild-type (Chd7^+/+), heterozygous (Chd7^Whi/+) and homozygous (Chd7^Whi/Whi)) were used according to the standard protocol of the Transcriptome Analysis Laboratory (TAL), Goettingen.

Adding 1 ml TRizol Reagent (Invitrogen) the entire embryo was lysed in a rotor homogenisator for 5 minutes at 50 oscillations. Next, the homogenate was incubated for 5 minutes at room temperature. To remove insoluble material from the homogenate, samples were centrifuged at 12,000 x g for 10 minutes at 4 °C. The supernatant was transferred to a fresh tube and the sample incubated again for 5 minutes at room temperature. 200 µl chloroform was added, tubes were vigorously shaken by hand for 15 seconds, then incubated 5 minutes at room temperature. After a 15 minute centrifugation step at 12,000 x g at 4 °C, the upper aqueous phase containing the RNA was transferred to a fresh 2 ml tube. Next, the RNA was precipitated by adding 500 µl isopropanol and 1 µl GlycoBlue. Samples were vortexed and incubated at -20 °C for 2 hours or overnight. The samples were centrifuged at 12,000 x g for 30 minutes at 4 °C. The supernatant was removed and the RNA pellet was washed with 1 ml 75 % ethanol. Samples were centrifuged at 12,000 x g for 5 minutes at 4 °C and the supernatant discarded. In case of a DNase

46

treatment a repetition of the wash step is not necessary. The RNA pellet was air-dried and dissolved in 25 µl DEPC. The concentration of the RNA was determined using a photo spectrometer (Eppendorf) (compare 2.2.2) and stored at 80 °C.